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A genetic hierarchy establishes mitogenic signalling and mitotic competence in the renal tubules of Drosophila

Vikram Sudarsan^1,*, Sara Pasalodos-Sanchez^1,*, Susan Wan¹, Alexandra Gampel² and Helen Skaer^1,*,

¹ Centre for Developmental Genetics, BMS, University of Sheffield, UK, S10 2TN
² Department of Biochemistry, University of Bristol, UK, BS8 1TD
^* Present address: Department of Zoology, Downing Street, Cambridge, UK, CB2 3EJ

View larger version (144K): [in a new window]   Fig. 1. Cell division in the Malpighian tubules. Tubules dissected from wild-type (A,B) and topco mutant (C,D) embryos stained for the incorporation of BrdU to mark S phase. The third postblastodermal division (cycle 16) occurs in a domain on the posterior side of the tubules (arrow in A) and takes place normally in mutant tubules (arrow in C). Only cells in the distal part of the tubules enter cycle 17 (arrow in B). This and subsequent cycles fail in tubules from topco mutants (arrow in D). (E,F) Mutant embryos stained for Cut. The tubules of topco mutant embryos are very small (arrow in E) but are partially rescued in embryos that also carry a deficiency uncovering reaper and hid (tubule cells arrow in F).

View larger version (80K): [in a new window]   Fig. 2. Rhomboid expression in the tip cell lineage. (A,B) Embryos of the genotype asense-Gal4 x UAS-nlacZ and stained for ß-galactosidase reveal expression in the tip mother cell at early stage 11 (arrowhead in A) and in both its daughters at stage 12 (arrowheads mark the tip and sibling cell in B). (C,D) Tubules from A37 embryos in which ß-gal (green) is expressed under the regulation of the neuromusculin enhancer, stained with an antibody against Rhomboid (red). At stage 11, ß-gal (arrowheads indicate tip mother cells) and Rho (arrows) are weakly expressed in the tip mother cells (C) and more strongly in both daughters from stage 12 onwards (D, stage 13: arrowheads, ß-gal; arrows, Rho). (E,F) In numb mutants (E) or if an activated N construct, Nintra is expressed in the tip mother cell using asense-Gal4 (F), the TC is lost. Instead, two SCs are specified and remain fully integrated in the tubule epithelium [arrowheads and marked by ß-gal (green) in E]. Rho (red) is expressed in both SCs. (G,H) In embryos mutant for the proneural (G) or neurogenic (H) genes TC specification is disrupted. (G) In embryos lacking the AS-C, the tubules (labelled for Cut in green) develop without tip and sibling cells and lack Rho, even though it is expressed in the midline (arrow in the inset). (H) In embryos mutant for N, the whole proneural cluster develops into TCs (eight to 12 cells in each tubule, labelled for Ac in green) and all these cells express Rho (red). aMT, anterior; pMT, posterior Malpighian tubule.

View larger version (100K): [in a new window]   Fig. 3. Notch is necessary, but not sufficient, for tubule cell division. Embryos stained for Cut reveal that the tubules of N mutant embryos (B) are shorter than wild type (A) (arrows). This results from a dramatic reduction in cell division from M17, demonstrated by failure to incorporate BrdU. Compare stained cells in the distal region of dissected tubules from stage 12 wild type (arrowheads in C) with the absence of incorporation in tubules from N mutant embryos of the same age (arrowhead in D). Driving the expression of activated Notch (hs-Nintra) from 5-10.5 hours in the tubules fails to rescue cell division and tubule growth in AS-C–/– [Df(svr)] embryos (compare arrows in E,F). Ubiquitous expression of an activated EGF receptor (UAS-top) also fails to rescue the tubule phenotype in either N (arrow in G) or AS-C–/– embryos (arrow in H). A,B,E-H are stained for Cut.

View larger version (82K): [in a new window]   Fig. 4. svp expression in the proneural cluster. (A) BrdU incorporation (purple) in the tubules of stage 12 embryos expressing svp-lacZ (green) demonstrates that the cells in S phase also express svp (overlay; white). (B,C) Kr staining (purple) marks the tubule cells and is more strongly expressed in the tip mother cell at early stage 11(arrowhead, B). Slightly later, Kr expression is lost from cells surrounding the tip mother cell (C). svp-lacZ is expressed in wild-type embryos before the tip mother cell divides (B, in C the tip mother cell is in anaphase) and therefore before EGF receptor-driven cell divisions. (D) In AS-C–/– [Df(svr)] embryos, there is no PNC and svp-lacZ is not expressed during stage 11 in the tubules. (E) In N55e11 embryos, supernumerary TCs, which develop from the PNC, express high levels of Kr and svp-lacZ, while other tubule cells express neither. (F) In embryos mutant for EGFR (topCO), svp-lacZ is still expressed in a cluster of tubule cells at stage 11, as in wild type (B).

View larger version (90K): [in a new window]   Fig. 5. pntP2 expression in the tubules is regulated by Wg. The expression of pnt is driven by two promoters, P1 and P2, resulting in the expression of the isoforms PntP1 and PntP2 (Scholz et al., 1993). The isoform expressed more strongly in the tubules is PntP2. (A) A stage 10 (4.75 hours) pntP2-lacZ embryo stained for Ac (green) and ß-gal (purple, overlay; white) shows that pntP2 is expressed in a wider domain than the PNC. (B-E) In situ hybridisation for pntP2, showing expression on the posterior side of the tubules (arrow in B, late stage 10; C, stage 11; arrowheads indicate the anterior side). (D) In wgcx4 mutant embryos, pntP2 expression fails in the tubules but is unaltered in the mesoderm (m; posterior side of tubule is indicated with an arrow, early stage 11). (E) Ectopic expression of wg by activating hs-wg from 3.5 to 4.5 hours results in elevated levels of pntP2 expression throughout the tubules (arrow indicates the posterior and arrowhead the anterior side of the tubule, late stage 10). e, epidermis; m, mesoderm; AMT, anterior Malpighian tubules; HG, hind gut; MTs, Malpighian tubules; PMG, posterior midgut; PMT, posterior Malpighian tubules.

View larger version (13K): [in a new window]   Fig. 6. Model for the establishment of cell fate in the MTs. Wg signalling activates the expression of pntP2 in a subset of tubule cells (brown), and is required within this domain to establish proneural gene expression in the PNC (represented here by ac, dark brown). Moderate levels of Ac induce svp expression in the PNC (grey) but, as Ac strengthens in the tip mother cell, a threshold for the initiation of rho expression is reached (blue). The tip mother cell divides to produce the TC and SC (blue). As ac expression is lost from the SC by stage 12, the maintenance of rho expression is later independent of Ac and may depend on a positive feedback loop. The svp- and pntP2-positive cells, in grey, are descended from the PNC and are competent to respond to EGF signalling. They divide in response to Spitz secreted by the tip and sibling cells.